Composition and method for self-assembly and mineralization of peptide amphiphiles

ABSTRACT

The present invention is directed to a composition useful for making homogeneously mineralized self assembled peptide-amphiphile nanofibers and nanofiber gels. The composition is generally a solution comprised of a positively or negatively charged peptide-amphiphile and a like signed ion from the mineral. Mixing this solution with a second solution containing a dissolved counter-ion of the mineral and/or a second oppositely charged peptide amphiphile, results in the rapid self assembly of the peptide-amphiphiles into a nanofiber gel and templated mineralization of the ions. Templated mineralization of the initially dissolved mineral cations and anions in the mixture occurs with preferential orientation of the mineral crystals along the fiber surfaces within the nanofiber gel. One advantage of the present invention is that it results in homogenous growth of the mineral throughout the nanofiber gel. Another advantage of the present invention is that the nanofiber gel formation and mineralization reactions occur in a single mixing step and under substantially neutral or physiological pH conditions. These homogeneous nanostructured composite materials are useful for medical applications especially the regeneration of damaged bone in mammals. This invention is directed to the synthesis of peptide-amphiphiles with more than one amphiphilic moment and to supramolecular compositions comprised of such multi-dimensional peptide-amphiphiles. Supramolecular compositions can be formed by self assembly of multi-dimensional peptide-amphiphiles by mixing them with a solution comprising a monovalent cation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/425,536 filed Nov. 12, 2002, entitled SELF ASSEMBLY OFMULTIDIMENSIONAL PEPTIDE AMPHIPHILES and the benefit of U.S. ProvisionalApplication No. 60/425,689 filed Nov. 12, 2002, entitled COMPOSITION ANDMETHOD FOR SIMULTANEOUS SELF-ASSEMBLY AND MINERALIZATION OFPEPTIDE-AMPHIPHILES and the benefit of International Application No.PCT/US03/04779 filed Feb. 18, 2003, entitled “SELF-ASSEMBLY OFPEPTIDE-AMPHIPHILE NANOFIBERS UNDER PHYSIOLOGICAL CONDITIONS”, thecontents of which are incorporate herein by reference in their entirety.

GOVERNMENT INTEREST

The United States government may have certain rights to this inventionpursuant to Grant Nos. N00014-99-1-0239/P00001, DMR-9996253, andDE-FGO2-00ER45810 from respectively, the Office of Naval Research, theNational Science Foundation, and the Department of Energy toNorthwestern University.

BACKGROUND OF THE INVENTION

Self-assembled gels composed of peptide-amphiphile nanofibers have beendescribed as being useful in the templated mineralization ofhydroxyapatite. Peptide-amphiphiles enriched with negatively chargedamino acids such as phosphoserine and aspartic acid can self assembleinto nanofibers and induce hydroxyapatite crystals to grow on thesurface of the nanofiber as described by Hartgerink et al., Science,294, 1683-1688, (2001). In addition to providing sites forhydroxyapatite crystal nucleation, the nanofibers also direct the growthof the hydroxyapatite crystals such that their c-axis is orientedparallel to the long axis of the nanofibers. The ability of thepeptide-amphiphile nanofibers to organize and direct the growth of thehydroxyapatite crystals is reminiscent on that observed between collagenfibrils and hydroxyapatite crystals in bone.

The directed growth of hydroxyapatite crystals within organizedpeptide-amphiphile matrices and scaffolds is an important step towardthe regeneration of mineralized materials like bone within the body.

While the preparation of oriented hydroxyapatite crystals on individualor small groups of nanofibers has been demonstrated, scaling the utilityof hydroxyapatite or other minerals in bundles of nanofibers or withingels comprising nanofibers maybe limited by non-homogeneousmineralization. Non-homogeneous mineralization of nanofiber bundles ornanofiber gels results in coating of the surface nanofibers of thebundle or gel by the mineral crystals. The formed surface crystalsinhibit further diffusion of mineral reagents into the interior of thenanofiber bundle or nanofiber gel and precludes formation of largerhomogenous composites. In practical applications such as boneregeneration, it would be desirable that hydroxyapatite crystal growthproceed uniformly throughout the nanofiber gel matrix.

A supramolecular assembly is a material in which the constituent unitsor building blocks of the assembly are molecules or molecularaggregates. The interaction of the units with each other, usually bynon-covalent bonding, determines the final shape and size of thesupramolecular assembly. An example of a supramolecular assembly foundin biological systems is α-hemolysin which is a seven protein aggregatewith a non-symmetric mushroom shape. The α-hemolysin aggregate has apore or channel that is about 16 Å in diameter, which runs parallel tothe aggregate's long axis. The aggressive human pathogen Staphylocuccusaureus uses the asymmetric nature of α-hemolysin to implant its steminto the hydrophobic compartment of cell membranes and the hydrophilicnature of the α-hemolysin's mushroom cap to stabilize it in theextracellular space. It is though α-hemolysin's pore channel that RNAmacromolecules from the Staphylocuccus aureus pathogen can invade humancells. Synthetic supramolecular assemblies could be designed andsynthesized to mimic the action of α-hemolysin's channel pore for drugdelivery or other cell therapies.

The amino acid sequence IKVAV (SEQ ID NO: 1) has been identified inother contexts as important for neuron growth and development. Selfassembly of peptide-amphiphiles with the IKVAV (SEQ ID NO: 1) sequencehave been reported. These peptide-amphiphiles may facilitate neurongrowth and development in supramolecular structures formed by thesepeptide-amphiphiles. One feature of peptide-amphiphiles having ahydrophobic alkyl tail and the IKVAV (SEQ ID NO: 1) amino acid sequencein the peptide head group is that peptide-amphiphile has more than oneamphiphilic moment. The peptide sequence of these and otherpeptide-amphiphiles can be further modified by covalent attachment ofligands or peptide sequences that can interact with various types ofcells. For example, the peptide sequence Arg-Gly-Asp (RGD) occurs infibronectin and has been found to play an important role inintegrin-mediated cell adhesion. Inclusion of the RGD peptide sequenceligand into a suitable peptide-amphiphile is expected to promote cellgrowth and direct templated mineralization of self assembledsupramolecular structures of such peptide-amphiphiles under the properconditions. Self assembled peptide-amphiphiles are known to direct themineralization of hydroxyapatite on the surfaces of nanofibers formedfrom these peptide-amphiphiles. The peptide portion of thesepeptide-amphiphiles can also comprise amino acid groups like cysteine,which are capable of forming disulfide bonds between adjacentpeptide-amphiphiles, and also glycine which provides flexibility to thepeptide portion of the molecule.

It will be appreciated by those skilled in the art that there is a needto be able to form self assembled supramolecular structures frompeptide-amphiphiles having more than one amphiphilic moment in order totake advantage of the unique cell growth, molecular transport, andtemplating functions that these and other related peptide sequencesprovide. It will also be appreciated that the self assembly occur inphysiologically benign conditions of temperature, ionic strength, andpH. For the foregoing reasons, there is a need in the art to makesupramolecular assemblies from multi-dimensional peptide-amphiphiles.

The present invention is directed to amphiphilic molecular compositionshaving more than one amphiphilic moment and also to supramolecularcomposition comprised of such amphiphilic molecules. More specifically,the present invention is directed to peptide-amphiphiles compositionshaving more than one amphiphilic moment and to supramolecularcompositions comprised of such peptide-amphiphiles which self assemblein the presence of cations.

Preferred embodiments of the present invention may be useful for cellgrowth, molecular transport, and templating functions, especially if theself assembly occurs under benign conditions.

Homogeneously, or substantially homogeneously, mineralized selfassembled peptide-amphiphile nanofibers are desirable. Homogenouslymineralized materials with the mineral crystals preferentially orientedby the self assembled peptide-amphiphile nanofibers are also desired.Finally, preparing such materials under substantially neutral orphysiological conditions is also desirable.

SUMMARY OF THE INVENTION

One embodiment of the present invention is directed to a composition formaterial formation on self assembled peptide-amphiphiles comprising atleast one ionically charged species of peptide-amphiphile and at leastone salt providing at least one ion from the material to be formed andhaving the same signed ionic charge as the peptide-amphiphile.Alternatively, the composition may be a solution of at least one speciesof peptide-amphiphile wherein the species of peptide-amphiphile chelatesone or more ions of the material to be formed. Charged and chelatingpeptide-amphiphiles may also be combined to form compositions for selfassembly and material formation.

The present invention is directed to a composition for materialformation on self assembled peptide-amphiphiles comprising at least oneionically charged species of peptide-amphiphile; and at least one saltproviding at least one ion from the material to be formed and having thesame signed ionic charge as the peptide-amphiphile.

The invention is also directed to a method of making materials on selfassembled peptide-amphiphiles, the method comprises preparing a firstsolution with at least one ionically charged species ofpeptide-amphiphile and at least one salt providing at least one ion fromthe material and having the same signed ionic charge as thepeptide-amphiphile. A second solution is prepared with an ion from thematerial and having opposite signed ionic charge to thepeptide-amphiphile in the first solution. The first and second solutionsare mixed to cause self-assembly of the peptide amphiphile nanofibersand to form the material substantially on the surfaces of thepeptide-amphiphile nanofibers throughout the nanofiber gel.

The present invention is directed to a composition useful for makinghomogeneously mineralized self assembled peptide-amphiphile nanofibersand nanofiber gels. The composition is generally a first solutioncomprised of a soluble positively or negatively chargedpeptide-amphiphile and a soluble salt containing an ion from themineral. The sign of the charge on the ion in the solution is the sameas sign of the charge on the peptide-amphiphile. Mixing this firstsolution with a second solution containing a dissolved counter-ion ofthe mineral and/or a second oppositely charged peptide amphiphile,results in the rapid self assembly of the peptide-amphiphiles into ananofiber gel with templated mineralization on the nanofibers of thesalt ions from the solution. Templated mineralization of the initiallydissolved mineral cations and anions in the mixture can occur withpreferential orientation of the mineral crystals along the fibersurfaces within the nanofiber gel.

One advantage of the present invention is that it results in homogenousgrowth of the mineral throughout the nanofiber gel. Another advantage ofthe present invention is that the nanofiber gel formation andmineralization reactions occur in a single mixing step and can occurunder substantially neutral or physiological conditions. Thesehomogeneous nanostructured composite materials are useful for medicalapplications especially the regeneration of damaged bone or teeth inmammals. Non-medical applications of the present invention include themanufacture or coating of hard surfaces on substrates.

In another embodiment of the invention, the composition comprises one ormore peptide-amphiphile species having different peptide sequences.

BRIEF DESCRIPTION OF THE FIGURES

Various aspects and applications of the present invention will becomeapparent to the skilled artisan upon consideration of the briefdescription of the figures and the detailed description of the inventionwhich follows.

FIG. 1 illustrates the chemical structure of a peptide-amphiphileC₁₅H₃₁C(O)-CCCCGGGS(P)RGD-COOH (SEQ ID NO: 2).

FIG. 2 illustrates a transmission electron micrograph (TEM) of ahomogeneously mineralized nanofiber gel sample with orientedhydroxyapatite mineral growth.

FIG. 3 is a schematic drawing of the chemical structures of thepeptide-amphiphiles C₁₅H₃₁C(O)-CCCCGGGEIKVAV-COOH (SEQ ID NO: 3),Molecule 1, and C₁₅H₃₁C(O)-CCCCGGGEIKVAV-NH₂ (SEQ ID NO: 3), Molecule 2.

FIG. 4 is a TEM micrograph of the negatively charged peptide-amphiphilenanofibers of molecule 1 self-assembled in the presence of KCl.

FIG. 5 is a schematic drawing of the chemical structure of thepeptide-amphiphile C₁₅H₃₁C(O)-CCCCRFEFRFEFR-NH₂ illustrating theimportant groups of the molecule as well as a representation of themagnitude and direction of two of the amphiphilic moments in themolecule. For illustrative purposes, Region 1 may be an alkyl group thatis covalently bonded to Region 2, which may be divided further intosections 2A and 2B, Regions 1 and 2 together define a first amphiphilicmoment of the molecule. The molecule may further comprise a secondamphiphilic moment defined by Regions 2A and 2B, wherein Region 2Bcomprises the polar and non-polar amino acids labeled 3, 4, 5 and 6.

DETAILED DESCRIPTION OF THE INVENTION

As used throughout the specification, the following terms shall have thefollowing meanings, unless the context clearly indicates otherwise. Ananofiber is defined as a cylindrical micelle comprising self-assembledpeptide-amphiphiles. Examples of such nanofibers with a single speciesof peptide-amphiphile are described in Science, 294, 1684, (2001). Ananofiber gel comprises a colloidal suspension of self assembledpeptide-amphiphile nanofibers and a liquid. The nanofiber gel behaves asan elastic solid and retains its shape. Mineralization is acrystallization process used to describe the nucleation and growth ofmineral crystals on the surface of a nanofiber or on the surfaces ofnanofibers throughout a nanofiber gel.

Although the present invention will be described in considerable detailwith respect to template mediated mineralization of hydroxyapatite onself assembled peptide-amphiphile nanofibers ofC₁₅H₃₁C(O)-CCCCGGGS(P)RGD-COOH (SEQ ID NO: 2), it is not intended to belimited to this system. Other materials, minerals, biominerals, magneticmaterials, conductive materials, and crystals, for example:fluoroapatite, calcium oxalate, calcite, tin hydrogen phosphate, ironoxides, iron hydroxides, and various iron oxyhydroxides, (Fe₂O₃, Fe₃O₄),TiO₂, ZnO, and versions of these materials containing substitutions ofthe ions, vacancies, or interstitial ions, may be nucleated and grown bythe practice of this invention. The invention is not limited by the sizeof the crystals or crystallites formed on the self assembledpeptide-amphiphiles. The formed crystals may be semi-crystalline aswell. Numerous positively and negatively charged peptide amphiphilespecies may be used in this invention, for exampleC₁₅H₃₁C(O)-CCCCGGGS(A)RGD-COOH (SEQ ID NO: 5), as well as those listedin Table 1 and Table 2. Although the present invention is described withrespect to aqueous solutions, addition of other liquids or solvents likeethanol to the solution is not precluded in the practice of thisinvention. The invention may also be practiced by adding an effectiveamount of the peptide-amphiphile and salts as powders to a surgicalsite, for example, where fluids containing ions needed for gelation andmineralization may be found.

The peptide-amphiphiles and their self assembled nanofibers may promoteadhesion and growth of cells on their surfaces. For example, the celladhesion ligand RGD has been found in other contexts to play animportant role in integrin-mediated cell adhesion. Peptide-amphiphilespecies with acidic amino acids and an amino acid with the RGD ligandcould be used to mediate cell adhesion to the peptide-amphiphiles, theirself assembled nanofibers, or nanofiber gels. The amino acid sequenceIKVAV (SEQ ID NO: 1) has been identified in other contexts as importantfor neuron growth and development. Accordingly, peptide-amphiphilespecies with acidic amino acids and the IKVAV (SEQ ID NO: 1) sequencecould be used in the practice of this invention to mediate neuron growthto the peptide-amphiphiles, their self assembled nanofibers, ornanofiber gels. The amino acid sequence YIGSR (SEQ ID NO: 6) has beenidentified in other contexts as important in for promotingcell-substrate adhesion among nerve cells also to play a role in axonguidance. Accordingly, peptide-amphiphile species with acidic aminoacids and the YIGSR (SEQ ID NO: 6) sequence could be used in thepractice of this invention to promote cell-substrate adhesion amongnerve cells to the peptide-amphiphiles, their self assembled nanofibers,or their nanofiber gels. For example in dentin, the phosphophorynprotein family contains numerous repeats of the amino acid sequencesAsp-Ser(P)-Ser(P) and Ser(P)-Asp. These massively phosphorylatedproteins are suspected to play an important role in hydroxyapatitemineralization. Accordingly, phosphoserine residues can be incorporatedinto the peptide sequence which, after self assembly, allows the fiberto display a highly phosphorylated surface equivalent to that presentedby a long peptide segment. This, in part, captures the repetitiveorganization of phosphate groups found in phosphophoryn proteins.

In one embodiment a composition useful in the self assembly andmineralization of peptide-amphiphiles comprises a first solution of atleast one negatively charged species of peptide-amphiphile and a solublesalt providing an anion of the mineral. The magnitude of the charges onthe peptide amphiphile and anion do not have to be the same. Thepeptide-amphiphile is prepared using standard solid phase chemistryknown to those skilled in the art. The dissolved anion may be obtainedfrom a soluble salt or salts comprising the mineral. Alternatively, themineral anion is formed by reaction known to those skilled in the art ofthe salts, for example with the pH adjustment of the solution, to yieldthe anion of the mineral. In cases where the mineral has more than oneanion, a mixture of salts comprising the anions of the mineral may beused. In a preferred embodiment NaH₂PO₄ is the source of phosphate ionfor the formation of hydroxyapatite. A second solution comprising one ormore cations of the mineral obtained from soluble salt or salts is mixedwith the first solution resulting in the self assembly of thepeptide-amphiphiles into a nanofiber gel. The second solution mayoptionally contain one or more positively charged peptide-amphiphiles.Templated mineralization of the cations and anions in the mixture occurswithin the nanofiber gel formed from the peptide-amphiphiles.

In another embodiment, a composition useful in the self assembly andmineralization of peptide-amphiphiles comprises a first solution of atleast one positively charged species of peptide-amphiphile and a solublesalt providing a cation of the mineral. The magnitude of the charges onthe peptide amphiphile and cation do not have to be the same. Thepeptide-amphiphile is prepared using standard solid phase chemistryknown to those skilled in the art. The dissolved cation may be obtainedfrom a soluble salt or salts comprising the mineral. Alternatively, themineral cation is formed by reaction of salts with the pH adjustedsolution to yield the cation of the mineral. In cases where the mineralhas more than one cation, a mixture of salts comprising the cations ofthe mineral may be used. A second solution comprising one or more anionsof the mineral obtained from soluble salt or salts is mixed with thefirst solution resulting in the self assembly of the peptide-amphiphilesinto a nanofiber gel. The second solution may optionally contain one ormore negatively charged peptide-amphiphile. Templated mineralization ofthe cations and anions in the mixture occurs within the nanofiber gelformed by the peptide-amphiphiles.

In cases where the mineral has more than one cation, a mixture of saltscomprising the cations may be mixed with the first solution to yield thehomogeneous nanostructured material. The salt may be organic, inorganic,or a peptide-amphiphile. Examples of such cations obtained from saltsand useful in the practice of this invention include but are not limitedto NH₄ ⁺, Na⁺, Al⁺³, Fe⁺³, Mg⁺², Fe⁺², Ca⁺², Zn⁺², Cu⁺², Gd⁺³ andmixtures of these ions. Peptide amphiphiles with a positive charge maybe considered as cations for the practice of this invention. Examples ofanions useful in the practice of this invention include but are notlimited to PO₄ ⁻³, AsO₄ ⁻³, CO₃ ⁻², OH⁻, C₂O₄ ⁻² silicates, sulfates andmixtures of these and other anions known to those skilled in the art.Peptide amphiphiles with a negative charge may also be considered asanions useful in the practice of this invention.

In another embodiment, the compositions can further comprise mixture ofpeptide-amphiphiles having the same signed ionic charge, but havingdifferent peptide sequences, functional groups, or magnitude of ioniccharge. Acidic groups on poly-peptide substrates plays a key role inbiomineralization processes. Phosphorylated groups are particularlypreferred in this regard.

In another embodiment one or more of the peptide amphiphiles chelates anion of the material to be formed. The chelating peptide-amphiphile maybe neutral or ionically charged. The peptide amphiphile chelating theion is then mixed with suitable ions or other peptides to formself-assembled nanofiber gels.

Notwithstanding embodiments provided above, broader aspects of thepresent invention include a peptide amphiphile composition having ahydrophobic or lyophobic component and a lyophilic peptide orpeptide-like component. In various preferred embodiments, thehydrophobic component of such a composition is of sufficient length toprovide amphiphilic behavior and micelle formation in water or anotherpolar solvent system. Typically, such a component is a C₆ or greaterhydrocarbon moiety, although other hydrophobic, hydrocarbon and/or alkylcomponents could be used as would be well-known to those skilled in theart to provide similar functional effect. Examples of such groupsinclude but are not limited to arachidonyl, various length vinylicgroups containing substituted with hydrogen or halogens such asfluorine, chlorine, bromine and iodine; acetylenic, diacetylenic andother acetylenic oligomers; various length alkene and isoprene groupssubstituted with hydrogen or halogens such as fluorine, chlorine,bromine and iodine. Regardless, the peptide component of such acomposition can include the aforementioned RGD, IKVAV (SEQ ID NO: 1), orother sequences found especially useful for the nanofiber mineralizationdescribed herein.

Preferred peptide components of such compositions can also include aphosphoryl-functionalized residue or sequence, as described above.Inclusion of a phosphoserine residue has been found especially usefulfor hydroxyapatite mineralization. Other embodiments can include aphosphotyrosine residue. The peptide component of such compositions alsoinclude a residue or sequence capable of promoting intermolecularbonding and structural stability of the nanofibers available from suchcompositions. A sequence of cysteine residues can be used with goodeffect, providing for the facile intermolecular oxidation/reduction ofthe thiol functionalities.

Peptide components of this invention preferably comprisenaturally-occurring amino acids. However, incorporation of artificialamino acids such as beta or gamma amino acids and those containingnon-natural side chains, and/or other similar monomers such ashydroxyacids are also contemplated, with the effect that thecorresponding component is peptide-like in this respect. Accordingly,such artificial amino acids, hydroxyacids or monomers can be used tomeet the phosphorylation and/or intermolecular bonding objectivesdescribed above.

Various aspects of the present invention can be described with referenceto the peptide amphiphile illustrated in FIG. 1. Consistent with broaderaspects of this invention, other peptide-amphiphiles, for example thoselisted in Table 1, may be used for the self-assembly of fibrouscylindrical micelles.

TABLE 1 PA N-terminus Peptide (N to C) C-terminus 1 C16 CCCCGGGS(P)RGDCOOH (SEQ ID NO:2) 2 C16 CCCCGGGS(P) COOH (SEQ ID NO:7) 3 HCCCCGGGS(P)RGD COOH (SEQ ID NO:2) 4 C10 CCCCGGGS(P)RGD COOH (SEQ IDNO:2) 5 C6 CCCCGGGS(P)RGD COOH (SEQ ID NO:2) 6 C10 GGGS(P)RGD COOH (SEQID NO:8) 7 C16 GGGS(P)RGD COOH (SEQ ID NO:8) 8 C16 AAAAGGGS(P)RGD COOH(SEQ ID NO:9) 9 C10 AAAAGGGS(P)RGD COOH (SEQ ID NO:9) 10 C16CCCCGGGS(P)KGE COOH (SEQ ID NO:10) 11 C10 AAAAGGGS(P)KGE COOH (SEQ IDNO:11) 12 C16 AAAAGGGS(P)KGE COOH (SEQ ID NO:11) 13 C22 CCCCGGGS(P)RGDCOOH (SEQ ID NO:2) 14 C16 CCCCGGGSRGD COOH (SEQ ID NO:12) 15 C16CCCCGGGEIKVAV COOH (SEQ ID NO:3) 16 C16 CCCCGGGS(P)RGDS COOH (SEQ IDNO:13) 17 C16 CCCCGGGSS(P)D(S(P)D COOH (SEQ ID NO:14)

It should be noted that within the system examined, PAs 3 and 5 do notexhibit micelle formation, demonstrating a certain degree ofhydrophobicity required for self-assembly of such compositions into thenanofibers of this invention. Depending upon desired cell or mineralgrowth, a phosphorylated moiety may not be required (see PAs 14 and 15).As discussed above, cellular adhesion or interaction is promoted by aparticular sequence of the peptide components. With reference to PA's10-12 and 15, a non-RGD sequence can be utilized depending upon cellulartarget. In particular, the IKVAV (SEQ ID NO: 1) sequence has beenidentified in other contexts as important for neuron growth anddevelopment. Accordingly the amphiphile compositions of this inventioncan include a peptide component having such a sequence for correspondinguse. Lastly, with respect to Table 1, it is noted that several PAcompositions do not include cysteine residues: while such a peptidesequence can be used to enhance intermolecular nanofiber stability, itis not required for micelle formation in the first instance.

In part, the present invention also provides for a system including anaqueous solution of one or more of the amphiphile compositions describedherein, and a factor or reagent sufficient to induce gelation underphysiological conditions. Such gelation and/or self-assembly of variousPA compositions into cylindrical micelle nanofibers can be achievedunder substantially neutral pH conditions through drying, introductionof monovalent, divalent, or higher valency ions and/or the combinationof differently charged amphiphiles. The approach of using differentlycharged amphiphiles can also be utilized to deliver in the selfassembling nanofibrous system two or more bioactive molecules, eachbearing different charges and this way combining the gelation technologywith the delivery of multiple biological signals. Such facile factors,as described more fully below and in several of the following examples,can extend the system and/or methodology of this invention to a varietyof medical applications. These and other aspects of the presentinvention can be described with reference to the peptide-amphiphile, PA,compositions provided in Table 2, and with further reference to FIG. 1and Table 1.

TABLE 2 Net Charge at PA N-terminus Peptide (N to C) C-terminus pH 7 18C16 CCCCGGGS(P)RGD COOH −3 (SEQ ID NO:2) 19 C16 AAAAGGGS(P)RGD COOH −3(SEQ ID NO:9) 20 C10 AAAAGGGS(P)RGD COOH −3 (SEQ ID NO:9) 21 C16CCCCGGGSRGD COOH −1 (SEQ ID NO:12) 22 C16 CCCCGGGEIKVAV COOH −1 (SEQ IDNO:3) 23 C16 CCCCGGGKIKVAV COOH +1 (SEQ ID NO:15)

In another embodiment of the invention, the degree of mineralization orcrystallization is controlled. By modifying the degree ofcrystallization, control of the physical properties of thepeptide-amphiphile mineral composite is achieved. The method comprisesaging the mixture of the first and second solutions to control theextent of the mineralization and crystal growth reaction. Crystal growthrequires, among other variables, control of the temperature and contacttime of the mixture containing the cations and anions with the nanofibergel.

As stated above, the amphiphile composition(s) of such a system mayinclude a peptide component having residues capable of intermolecularcross-linking. The thiol moieties of cysteine residues can be used forintermolecular disulfide bond formation through introduction of asuitable oxidizing agent or under physiological conditions. Converselysuch bonds can be cleaved by a reducing agent introduced into the systemor under reducing conditions. The concentration of cysteine residues canalso be varied to control the chemical and/or biological stability ofthe nanofibrous system and therefore control the rate of therapeuticdelivery or release of cells or other beneficial agent, using aneffective amount of the nanofibers as the carriers. Furthermore, enzymescould be incorporated in the nanofibers to control biodegradation ratethrough hydrolysis of the disulfide bonds. Such degradation and/or theconcentration of the cysteine residues can be utilized in a variety oftissue engineering contexts.

The ability of various peptide sequences in the peptide-amphiphiles topromote bone, tissue, or nerve growth may make systems of self assemblednanofibers useful in a number of different potential application.Specific applications include the delivery of therapeutics as well asbiomedical and tissue engineering. As a self-supporting gel, it may haveapplications as a mineralizable bone-defect filler.

The assembly in the presence of biological ions such as Ca²⁺ may makethe homogeneously mineralized material herein described particularlyvaluable for in situ and in vivo applications. It may also be used as abiological coating for orthopedic implants. These applications couldfind particularly valuable use in addressing medical problems such asosteooncology, congenital bone and tooth defects, osteoporosis,synthetic teeth, and dental implants.

The self-assembled peptide amphiphiles described in this disclosure aremodifications of those originally described by Hartgerink, et al. (Seee.g., J. D. Hartgerink, E. Beniash and S. I. Stupp, Science 294,1683-1688, 2001), which is hereby incorporated in its entirety byreference thereto and the synthetic schemes set forth therein applyactually as well to the present invention.

Self-assembly and/or gelation under physiological conditions raisesnumerous implication regarding the end-use application and effect. Apeptide-amphiphile mixture makes available a system for the formation ofmicellular nanofibers in an aqueous environment at neutral and/orphysiological pH conditions. Such a combination can be used to assemblenanofibers with a range of chemical groups or amino acids providing avariety of chemical or biological signals for corresponding celladhesion, yielding enhanced properties with respect to tissueengineering or regenerative applications. It is contemplated that, aloneor in conjunction with the other factors discussed herein, thatpreferred medical or therapeutic embodiments of such a system can beutilized. Furthermore, although the invention will be described indetail with respect to aqueous solution, the presence of non-aqueousliquids in the solution, like ethanol, will not limit the scope of theinvention. Similarly, use of the terms hydrophobic and hydrophilic todescribes the interaction of the multi-dimensional amphiphiles withwater are construed to be equivalent to lyophobic and lyophilic forinteraction of the multidimensional amphiphiles with non-aqueousliquids.

The present invention is directed to amphiphilic molecular compositionshaving more than one amphiphilic moment and also to supramolecularcomposition comprised of such amphiphilic molecules. An amphiphilicmolecule with more than one amphiphilic moment is referred to as amulti-dimensional amphiphile. An example of such a molecule is shownschematically in FIG. 5. The multi-dimensional amphiphilic molecule hasa first chemical group or moiety, 1, covalently bonded to a secondchemical group or moiety. In FIG. 5 the second moiety is further dividedinto sections 2A and 2B. In FIG. 5, and for illustrative purposes only,the second chemical moiety is a peptide comprised of amino acids. Theamino acids may be, for example, naturally occurring amino acids,synthetic amino acids, β-amino acids, γ-amino acids, and or mixtures ofthese amino acids. The first and second moieties define a firstamphiphilic moment of the amphiphilic molecule. The direction andmagnitude of the first amphiphilic moment is along the axis of themolecule and is represented by divergent lines and labels lyophobic-1and lyophilic-1 in FIG. 5.

The second moiety of the molecule is further comprised of moietiescovalently bonded together that define a second amphiphilic moment ofthe molecule. In FIG. 5, for example, the second moiety is a peptidecomprised of cysteine amino acids (2A) and polar and non-polar aminoacids labeled 3, 4, 5, and 6. The amino acid moieties 4 and 5 are polarand substantially lyophilic because of the nature of the substituents.Examples of these substituents may be, acid groups, amine and amidegroups, phosphate groups, hydroxyl groups, and sulfate groups, andcarboxylic acid groups. The amino acid moieties 3 and 6 are the same inthis example, but they may be different, and are non-polar andsubstantially lyophobic because of the nature of the substituents.Examples of these substituents may be phenyl, methyl, or substitutedalkyl groups. The sequence of polar/lyophilic and nonpolar/lyophobicmoieties that make up the second lyophilic moiety in the moleculedefines a second amphiphilic moment in the molecule. The secondamphiphilic moment is not parallel to the first amphiphilic moment ofthe molecule. In FIG. 5 the direction and magnitude of the secondamphiphilic moment lies or is oriented across the first amphiphilicmoment of the molecule; the two moments are not parallel to each other.The second amphiphilic moment is represented by the smaller divergentlines and labels lyophobic-2 and lyophilic-2 in FIG. 5. A molecule withmore than one amphiphilic moment is termed a multi-dimensionalamphiphile; a molecule with more than one amphiphilic moment and havinga peptide is termed a multidimensional peptide-amphiphile.

The portion of the peptide sequence labeled 2A in FIG. 5 has cysteineamino acids capable of bonding together adjacent multidimensionalpeptide-amphiphiles in a self assembled nanofiber. In the moleculedepicted in FIG. 5, the cysteine amino acids may be replaced withglycine amino acids to provide flexibility to the peptide portion of themolecule. The cysteine amino acids may be replaced with other polar ornon-polar amino acids. Other synthetic amino acids, b-amino acids,g-amino acids with polar or non-polar substituents may be used in thepractice of this invention. Incorporation monomers such as hydroxyacidsare also contemplated, with the effect that the corresponding componentis peptide-like in this respect. The sequence of alternating polar andnon-polar moieties and more specifically the alternating polar andnon-polar amino acids may be varied and the invention is not limited todisclosed combinations. The peptide may contain the amino acid sequenceIKVAV (SEQ ID NO: 1). Other examples of peptides with alternatinghydrophobic and hydrophilic amino acids, include but are not limited to:YQYQYQ (SEQ ID NO: 16); AQAQAQ (SEQ ID NO: 17); YQAQYQAQ (SEQ ID NO:18); RADARADA (SEQ ID NO: 19); HNHNHN (SEQ ID NO: 20); HNHQHNQH (SEQ IDNO: 21).

This invention is more specifically directed to the synthesis ofpeptide-amphiphiles with more than one amphiphilic moment and tosupramolecular compositions comprised of such multi-dimensionalpeptide-amphiphiles. These supramolecular compositions can be formed byself assembly of multi-dimensional peptide-amphiphiles by mixing themwith a solution comprising a cation. In a preferred embodimentmonovalent cations are used to induce self assembly of themultidimensional peptide-amphiphiles. Examples of such cations includebut are not limited to Na⁺, K⁺, or RNH₃ ⁺, where R is a hydrogen, aphenyl group, or an alkyl group.

In a preferred embodiment of the invention, a supramolecular compositionis formed by mixing multi-dimensional peptide-amphiphiles containing theIKVAV (SEQ ID NO: 1) amino acid sequence with a monovalent cation fromsalts such as NaCl and KCl. Examples of suitable multi-dimensionalpeptide amphiphiles are Molecule 1 and Molecule 2 illustrated in FIG. 3.The peptide-amphiphile has amino acids with moieties for covalentcoupling. Examples of such amino acids include but are not limited tocysteine. The peptide-amphiphile also has amino acids that provide aflexible linkage within the peptide portion of the molecule. Examples ofsuch amino acid moieties include but are not limited to gylcine.

More specifically, the peptide-amphiphiles of this invention contain ahydrophobic or lyophobic component of sufficient length to provideamphiphilic behavior and micelle formation in water or polar solutions.Typically, such a first moiety is a C₆ or greater hydrocarbon group,although other hydrocarbon and/or alkyl components could be used inplace of or bonded as a substituents onto the hydrocarbon group as wouldbe well known to those skilled in the art. Examples of such groupsinclude but are not limited to arachidonyl, various length vinylicgroups containing substituted with hydrogen or halogens such asfluorine, chlorine, bromine and iodine; acetylenic, diacetylenic andother acetylenic oligomers; various length alkene and isoprene groupssubstituted with hydrogen or halogens such as fluorine, chlorine,bromine and iodine.

The invention may also be practiced by adding an effective amount of thepeptide-amphiphile and salts as powders to a surgical site, for example,where fluids containing ions needed for gelation and mineralization maybe found.

The self assembly and gelation of peptide-amphiphiles like Molecule 1and Molecule 2 to form the supramolecular composition is triggered byaddition of monovalent cations into the peptide-amphiphile solution. Themonovalent salts provide an ionic environment that is believed to reducethe electrostatic repulsive force between peptideamphiphiles of the samepolarity. Examples of suitable monovalent cations include but are notlimited to Na⁺, K⁺, or RNH₃ ⁺. The monovalent cations in solution enablethe peptideamphiphiles to establish short range hydrophobic interactionsbetween the aliphatic tails of the molecules as well as the amphiphilicportions of the peptide sequence. Amphiphilic peptides were previouslyreported to self assemble into β-sheet based supramolecular structures(Aggeli et. al. 1977, and Holmes et al., 2000).

One advantage of the present invention is that the peptide amphiphilesself assemble to form fibers rather hollow tubes. Such fibers may besuitable for deliver or encapsulation of various cell therapies andprovide close surfaces for templated tissue, bone, or nerve growth. Thedelivery of an effective amount of such encapsulated therapeutics to apatient may be useful in the treatment of a variety of conditions. Thestructure of the peptide-amphiphile may be changed to create selfassembled structures having various pore sizes. Although the presentinvention will be described in considerable detail with respect to selfassembly of multi-dimensional peptide amphiphiles with the IKVAV (SEQ IDNO: 1) peptide and their use in promoting cell growth, it is notintended to be limited to this amino acid sequence or to cell growth.Other multi-dimensional peptide amphiphiles with alternating polar andnon-polar amino acids sequences may self assemble and direct the growthof tissues, materials, minerals, biominerals, magnetic materials,conductive and semiconductor materials, and crystals on their surfaces.Examples of such materials include but are not limited to fluoroapatite,calcium oxalate, calcite, tin hydrogen phosphate, iron oxides, ironhydroxides, and various iron oxyhydroxides, (Fe₂O₃, Fe₃O₄), TiO₂, ZnO.Versions of these materials containing substitutions of the ions,vacancies, or interstitial ions, may also be nucleated and grown by thepractice of this invention. The invention is not limited by the size ofthe crystals or crystallites formed on the self assembledpeptide-amphiphiles. The formed crystals may be semi-crystalline aswell.

Another difference between the peptide-amphiphiles in the presentinvention from known amphiphilic molecules is that the presentinvention's peptide-amphiphiles are two-dimensional amphiphiles. Thepeptide-amphiphiles of the present invention have two “amphiphilicmoments” oriented in different directions. One amphiphilic momentcoincides with or is parallel to the backbone axis of the molecule, thesecond amphiphilic moment is not parallel to the backbone of themolecule and is directed across the peptide sequence of the molecule.The alkyl tail moiety of the peptide-amphiphile is much more hydrophobicthan any moieties on the amino acids composing the peptide part of thepeptide-amphiphile. The amphiphilic moment along the backbone of thepeptide-amphiphile molecule is much stronger than the amphiphilicityacross the IKVAV (SEQ ID NO: 1) segment. The amphiphilicity in differentdirections is different; it is much stronger along the backbone of themolecule than along the sides of the amphiphilic peptide segment. Thismolecular design may serve as a prototype for other multi-dimensionalamphiphilic molecules, which may not include the peptide or alkylmoieties. In principle any molecule with two or more axes ofamphiphilicity may be described as a multi-dimensional amphiphile.Multi-dimensional amphiphiles can serve as the building blocks forsupramolecular assemblies and lead to the development of newsupramolecular structures that may find application in different fieldsof nanotechnology and biomedical applications.

Supramolecular compositions formed from self assembled multi-dimensionalamphiphiles may be administered to treat a patient. For example, thepatient may require assistance stimulating cell or nerve growth. Thetreatment comprises administering a multi-dimensional peptide-amphiphilecomposition having a cell growth peptide sequence within thepeptide-amphiphile to a site on the patient requiring treatment. Thesupramolecular composition may form using sodium or potassium ionsalready present in the patient. Alternatively a separate solutioncontaining monvalent ions may be administered to the patient to causethe formation of the supramolecular composition from themulti-dimensional peptide-amphiphile solution.

Synthetic supramolecular assemblies could also be designed andsynthesized with channels or pores for targeted delivery of drugs tospecific cells or organs. These supramolecular assemblies may providefor encapsulation of materials and molecules such as therapeutic drugs,cell therapies, cancer treatments, antibodies, magnetic colloids,conductive colloids, carbon nanotubes, and semiconductor colloids.

EXAMPLE 1

This example illustrates the components of a liquid composition and itsuse to form homogeneously distributed, and directionally orientatedhydroxyapatite crystals within a nanofiber gel comprised of selfassembled peptide-amphiphiles.

Peptide amphiphile C₁₅H₃₁C(O)-CCCCGGGS(P)RGD-COOH (SEQ ID NO: 2) (1) wasprepared using standard solid phase chemistry; its structure is shown inFIG. 1.

A liquid composition for the self assembly of (1) and mineralization ofhydroxyapatite was prepared by dissolving 20 millimoles NaH₂PO₄(Aldrich) into 200 microliters of (1) at a concentration of 10 mg/ml inwater. The pH of the solution was adjusted to 7.7.

The liquid composition containing (1) and NaH₂PO₄ was mixed with 40micromoles of CaCl₂ (Aldrich).

Self assembled gel comprising (1) was formed immediately upon additionof CaCl₂ to the liquid composition of the peptide-amphiphile (1)preloaded with a source phosphate anions from the NaH₂PO₄. The selfassembled gel was initially transparent but turned a white color afterabout 2 hours suggesting that the mineralization process had started.Samples of the gel after 2 hours, 1 day, and 5 days were mounted oncarbon-coated TEM grids. TEM studies of the 1 and 5 day samples show amulti-crystalline material composed of plate like crystals ˜5 nm thickand 50 to 100 nm long. The plate like crystals were similar to thoseobserved in the mineralization experiments on the pre-assembledtemplate. Electron diffraction of the material matches the diffractionpattern for hydroxyapatite. Both TEM images of the mineralized gel aswell as diffraction patterns suggest local orientation of thehydroxyapatite crystals in the self assembled gel. These data suggestthat the c-axis of the hydroxyapatite crystals is co-aligned with thenanofiber axis as shown in FIG. 2. This observation suggests that thepeptide-amphiphile nanofibrils control nucleation and direction of thecrystal growth.

The results of this example show that peptide-amphiphile organo-mineralcomposite materials may be manufactured in one step by adding metal ionsto a liquid composition of peptide-amphiphile pre-loaded with a sourceof phosphate anions. The example further illustrates that theself-assembly of the peptide-amphiphiles occurs upon addition of a metalion and that they later serve as a template for the directedmineralization of hydroxyapatite. This example further illustrates thatthe method for making the hydroxyapatite composites is useful forpreparing homogenous nanostructured composite materials.

EXAMPLE 2

In another example of this invention, the composition and methodconsists of using highly charged peptide amphiphile species (16carbonalkyl tail with a sequence like CCCCGGGSS(P)DS(P)D (SEQ ID NO: 14) witha −7 charge, for example) dissolved in a solution of negative ions(phosphate ions with a −3 charge), call this solution X. A secondsolution with a positively-charged peptide amphiphile species (such as16 carbon alkyl tail with a sequence like ACAAGGGKRGDS (SEQ ID NO:21)—an amine terminated PA at +1 charge) in a solution withpositively-charged ions, such as Ca²⁺; call this solution Y. In bothsolutions the peptide heads are charged and the structural element ofthe peptide can be varied, to give different charged peptide-amphiphilespecies, depending on the application.

The positive and negative peptide amphiphiles alone (no added salt ions)will gel each other, reaction 1, when mixed in the right ionicratios(1:7, (−):(+) in this instance), forming mixed peptide amphiphilenanofibers, theoretically composed of 7 positive peptide amphiphiles forevery 1 of the negative peptide amphiphiles. The positive peptideamphiphile solution Y may be gelled, reaction 2, with the negative ions(for example a solution containing phosphate ion PO₄ ⁻³). The negativepeptide amphiphile solution X will be gelled, reaction 3, with positiveions (for example a solution containing Ca⁺²). The positive peptideamphiphile does not gel, reaction 4, in positive ions (for example Ca⁻²)The negative PA does not gel, reaction 5, in negative ions (for examplephosphate ion PO₄ ⁻³). Mixing the positive and negative ions (calciumcation with phosphate anion) will make calcium phosphate mineral(reaction 6). When solution X is mixed with solution Y, a gel forms,reaction 7, very quickly. It is believed that mineral (calciumphosphates and possibly sodium chloride) is nucleated and grownintimately and substantially throughout the mixed-peptide amphiphilefibers. The gel formed may be the product of reactions 1, 2, 3, and 6,occurring in approximately the same time frame. The combination allowsus formation of a mineralized gel at physiologic pH. This examplefurther demonstrates that by using two distinct peptide-amphiphiles,different peptide sequences which might work well in concert withone-another (such as IKVAV (SEQ ID NO: 1) and YIGSR (SEQ ID NO: 6))might be simultaneously combined during the assembly and mineralizationprocess.

EXAMPLE 3

This example describes the synthesis of peptide-amphiphiles with morethan one amphiphilic moment, and describes the synthesis of asupramolecular composition comprised of self-assembled multi-dimensionalpeptide-amphiphiles. A supramolecular composition is formed by combiningmulti-dimensional amphiphiles containing the IKVAV (SEQ ID NO: 1) aminoacid sequence with monovalent salts such as NaCl and KCl.

Molecule 1, as shown in FIG. 3, is a peptide-amphiphile that containsthe amino acid sequence IKVAV (SEQ ID NO: 1) moiety with terminal endgroup —COOH; this sequence has been shown to promote axon outgrowth inneurons. Molecule 2, also shown in FIG. 3, is a peptide-amphiphile thatcontains the sequence IKVAV (SEQ ID NO: 1) moiety with the terminal endgroup—NH₂, which has similarly been shown to promote axon outgrowth inneurons. The two molecules dissolve in pH 7.5-adjusted water at aconcentration of about 10 mg/mL. Molecule 1 has a charge of (−1) andmolecule 2 has a charge of (+2) under these conditions. Aself-supporting gel forms on mixing of either of the peptide-amphiphilesolutions with 200 mM KCl or NaCl solutions. Examination of the gelsformed by these reactions by negative stain TEM shows that the gels arecomposed of nanofibers of the self assembled peptide-amphiphiles.

In all cases self assembled gels comprised of the nanofibers were formedsimilar to those described elsewhere (Hartgerink et al., 2001;Hartgerink et al., 2002). In contrast no self assembly or gel formationwas observed when other negatively or positively chargedpeptide-amphiphiles were exposed to the NaCl or the KCl atconcentrations up to 6 M. The fact that molecules 1 and 2 assemble inthe presence of the monovalent salts sets them apart from the othermolecules studied. Experiments with negatively charged molecules that donot contain amphiphilic peptide sequences show that the charge screeningby monovalent inorganic ions alone is not sufficient to inducepeptide-amphiphile self-assembly. The reason for this difference may bein the structure of these molecules. Both these molecules contain IKVAV(SEQ ID NO: 1) sequence at the c-terminus of the peptide segment. Thissequence is comprised of alternating extremely hydrophobic amino acids Iand V and more hydrophilic ones such as A and K. Since the side chainsof adjacent amino acids are located on opposite sides of the peptidebackbone, this sequence is amphiphilic. The molecules 1 and 2 may beconsidered as double or two dimensional amphiphiles; one moment ofamphiphilicity coinciding with the backbone axis of the molecule andamphiphilic peptide segment at c-terminus, and the second moment ofamphiphilicity directed across the amphiphilic peptide segment.Previously amphiphilic peptides have been shown to assemble into ribbonlike structures forming 3-D networks upon addition of monovalent salts(Zhang et al., 1995; Caplan et al., 2000). It was suggested that thefunction of inorganic ions in these systems is to screen chargedfunctional groups of the peptide that facilitates supramolecularassembly of amphiphilic peptides. It is believed that a similarmechanism is involved in the self assembly of peptide-amphiphilescontaining IKVAV (SEQ ID NO: 1) sequences in addition to the hydrophobicinteractions between the alkyl parts of the molecules.

Materials and Methods: Abbreviations: PA: peptide-amphiphile, TEM:transmission electron microscopy.

Chemicals: Except as noted below, all chemicals were purchased fromFisher or Aldrich and used as provided. Amino acid derivatives werepurchased from Applied BioSystems and NovaBiochem; derivatized resinsand O-Benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate(HBTU) were also purchased from NovaBiochem. All water used wasdeionized with a Millipore Milli-Q water purifier operating at aresistance of 18 MW.

Synthesis of the peptide-amphiphiles: The peptide-amphiphiles wereprepared on a 0.25 mmole scale using standard FMOC chemistry on anApplied Biosystems 733A automated peptide synthesizer. After the peptideportion of the molecules was prepared, the resin was removed from theautomated synthesizer and the N-terminus capped with a fatty acidcontaining 16 carbon atoms. The alkylation reaction was accomplishedusing 2 equivalents of the fatty acid, 2 equivalents HBTU and 6equivalents of n,n-diisopropylethylamine (DiEA) in dimethylformamide(DMF). The reaction was allowed to proceed for at least six hours afterwhich the reaction was monitored by ninhydrin. The alkylation reactionwas repeated until the ninhydrin test was negative. Only two couplingswere required in each case.

Cleavage and deprotection of the molecules was accomplished with amixture of trifluoroacetic acid (TFA) and triisopropylsilane (TIS) in aratio of 95:5 for three hours at room temperature. The cleavage mixtureand two subsequent TFA washings were filtered into a round bottom flask.The solution was roto-evaporated to a thick viscous solution. Thissolution was triturated with cold diethylether. The white precipitatewas collected by filtration, washed with copious cold ether and driedunder vacuum. The molecules were then dissolved in water at aconcentration of 10 mg/mL, adjusting the pH to improve solubility. Thesolution was initially acidic in both cases. In the case of molecule 1,the pH was raised to about pH 8 with 2M and 100 nM KOH, thenback-titrated to pH 7. In the case of molecule 2, the molecule was mostsoluble at low pH, but remained in solution when the pH was raised to 7using KOH. The molecules were characterized by ESI MS and were found tohave the expected molecular weight.

Transmission Electron Micrographs of samples of the supramolecularcompositions from the multi-dimensional peptide-amphiphiles, Molecule 1and Molecule 2, were prepared as follows. A small sample of thesupramolecular composition gel, prepared in bulk as described above, wassmeared onto a holey carbon coated TEM grid (Quantifoil). Negativestaining with PTA (phosphotungstic acid) was used in this study[Harris,1991#93]. In all cases electron microscopy was performed at anaccelerating voltage of 200 kV.

Various other amphiphile compositions of this invention can be preparedin analogous fashion, as would be known to those skilled in the art andaware thereof, using known procedures and synthetic techniques orstraight-forward modifications thereof depending upon a desiredamphiphile composition or peptide sequence.

All of the embodiments disclosed and claimed herein can be made andexecuted without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention.

While the principles of this invention have been described in connectionwith specific embodiments, it should be understood clearly that thesedescriptions are added only by way of example and are not intended tolimit, in any way, the scope of this invention. For instance, variouspeptide amphiphiles have been described in conjunction with specificresidues and corresponding cell adhesion, but other residues can be usedherewith to promote a particular cell adhesion and tissue growth on thenanostructures prepared therefrom. Likewise, while the present inventionhas been described as applicable to biometric material or tissueengineering, it is also contemplated that gels or related systems ofsuch peptide amphiphiles can be used as a delivery platform or carrierfor drugs, cells or other cellular or therapeutic material incorporatedtherein. Other advantages and features will become apparent from theclaims filed hereafter, with the scope of such claims to be determinedby their reasonable equivalents, as would be understood by those skilledin the art.

REFERENCES

-   -   Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature, 398,        495-498, (1999).    -   Braun, P. V.; Stupp, S. I. Materials Research Bulletin, 34,        463-469, (1999).    -   Hartgerink et al; Science, 294, 1684-1688, (2001).    -   Hartgerink et al, PNAS, 99(8), 5133-5138, (2002).    -   Preparation of self-assembling amphiphile for construction of        peptide secondary structures. G. B. Fields, M. V. Tirrell, U.S.        Pat. No. 6,096,863.    -   M. R. Caplan, P. N. Moore, S. G. Shang, R. D. Kamm, D. A.        Lauffenburger, Biomacromolecules; 1, 627, (2000).    -   A. L. Litvin, S. Valiyaveettil, D. L. Kaplan Process for        nucleation of ceramics and products thereof. U.S. Pat. No.        5,993,541    -   Xu, G. F. et al, Journal of the American Chemical Society 120,        11977-11985, (1998)

1. A method for the mineralization of nanofibers, the method comprising:preparing a first solution with at least one peptide amphiphilecomprising a C₆ or greater hydrocarbon component at its N-terminus and alyophilic peptide component; preparing a second solution of a mineralsalt; and mixing said first and second solutions to self-assemble saidpeptide amphiphiles into nanofibers and a nanofiber gel, wherein saidmineralization occurs from initially dissolved mineral cations andanions along the nanofibers surfaces, wherein said nanofibers arefibrous cylindrical micelles.
 2. The method claim 1 further comprising:aging the mixture of said first and second solutions to control the sizeand rate of growth of said minerals on the self-assembled peptideamphiphile nanofibers.
 3. The method of claim 1 further comprising:adjusting the pH of one of the solutions prior to mixing them together.4. The method of claim 1, wherein said mineral cations and anions areselected from the group consisting of hydroxyapatite, fluoroapatite,calcium oxalate, calcite, tin hydrogen phosphate, iron oxides, ironhydroxides, iron oxyhydroxyoxides, titanium dioxide, and zinc oxide. 5.The method of claim 1, wherein the peptide-amphiphile has a net negativecharge.
 6. The method of claim 4, wherein the mineral is hydroxyapatite.